JP2017165727A - 組成物及び皮膚外用剤 - Google Patents
組成物及び皮膚外用剤 Download PDFInfo
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- JP2017165727A JP2017165727A JP2017046598A JP2017046598A JP2017165727A JP 2017165727 A JP2017165727 A JP 2017165727A JP 2017046598 A JP2017046598 A JP 2017046598A JP 2017046598 A JP2017046598 A JP 2017046598A JP 2017165727 A JP2017165727 A JP 2017165727A
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Landscapes
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Abstract
【解決手段】バラ科(Rosaceae)サクラ属(Prunus)に属するサクラの抽出物と、酵母、麹菌及び乳酸菌のいずれか1種又は2種以上の抽出物又は酵母、麹菌及び乳酸菌のいずれか1種又は2種以上の微生物により産生される微生物由来発酵代謝物とを含有する組成物。
【選択図】なし
Description
また、本発明は、上記組成物を含む皮膚(頭皮も含む)外用剤又は飲食品であることを特徴とする。
本発明は、サクラの抽出物と、酵母、麹菌及び乳酸菌のいずれか1種又は2種以上の抽出物又は酵母、麹菌及び乳酸菌のいずれか1種又は2種以上の微生物により産生される微生物由来発酵代謝物とを含有する組成物である。
(i)サクラの抽出物の調製
サトザクラ(Prunus lannesiana)の花を乾燥、粉砕し、粉砕物18gに精製水900gを加え、80℃にて、2時間抽出後、濾過し、褐色透明のサクラ抽出物溶液1385gを得た(固形分濃度0.49%)。
(ii)微生物由来発酵代謝物
植物(サクラ)の花由来の酵母を、グルコース・ペプトンを含む溶液で、15℃で48時間培養し、培養液を濾過して得られる培養液を酵母抽出物溶液(固形分濃度0.51%)とした。
(iii)組成物の調製
サクラ抽出物溶液と酵母抽出物溶液を、1:1の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.40%)
(i)サクラの抽出物の調製
サトザクラの花を乾燥、粉砕し、粉砕物18gに精製水450gと1,3−ブチレングリコール450gを加え、80℃にて2時間抽出後、濾過し、褐色透明のサクラ抽出物溶液1365gを得た(固形分濃度0.45%)。
(ii)微生物由来発酵代謝物の調製
精製水800gに、清酒の製造過程で生じる酒粕160gを加え、80℃にて1時間抽出後、濾過し、淡黄色透明の酒粕抽出物溶液2490gを得た(固形濃度0.45%)
(iii)組成物の調製
サクラの抽出物と酒粕抽出物を、1:9の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.34%)。
(i)サクラの抽出物の調製
サトザクラの花を乾燥、粉砕し、粉砕物18gに精製水270gと1,3−ブチレングリコール630gを加え、80℃にて3時間抽出後、濾過し、褐色透明のサクラ抽出物溶液1350gを得た(固形分濃度0.39%)。
(ii)微生物由来発酵代謝物の調製
乳酸菌をMRS培地で、37℃で72時間液体培養し、培養液を濾過して得られる培養液を乳酸菌抽出物溶液(固形分濃度0.72%)とした。
(iii)組成物の調製
サクラ抽出物溶液と乳酸菌抽出物溶液を、1:2の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.51%)
(i)サクラの抽出物の調製
サトザクラに代えてヤマザクラを用いるほかは、製造例1の(i)と同様の方法により、褐色透明のサクラ抽出物溶液1350gを得た(固形分濃度0.43%)。
(ii)微生物由来発酵代謝物の調製
製造例1の(ii)と同様の方法で、酵母抽出物溶液を得た。
(iii)組成物の調製
サクラ抽出物溶液と酵母抽出物溶液を、1:1の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.37%)
(i)サクラの抽出物の調製
サトザクラに代えてヤマザクラを用いるほかは、製造例2の(i)と同様の方法により、褐色透明のサクラ抽出物溶液1355gを得た(固形分濃度0.41%)。
(ii)微生物由来発酵代謝物の調製
製造例2の(ii)と同様の方法により酒粕抽出物溶液を得た。
(iii)組成物の調製
サクラの抽出物溶液と酒粕抽出物溶液を、1:9の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.32%)。
(i)サクラの抽出物の調製
サトザクラに代えてヤマザクラを用いるほかは、製造例3の(i)と同様の方法により、褐色透明のサクラ抽出物溶液1345gを得た(固形分濃度0.35%)。
(ii)微生物由来発酵代謝物の調製
製造例3の(iii)と同様の方法にて、乳酸菌抽出物溶液を得た。
(iii)組成物の調製
サクラ抽出物溶液と乳酸菌抽出物溶液を、1:2の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.49%)
(i)サクラの抽出物の調製
サトザクラに代えてソメイヨシノ(Prunus×yedoensis)を用いるほかは、製造例1の(i)と同様の方法により、褐色透明のサクラ抽出物溶液1360gを得た(固形分濃度0.42%)。
(ii)微生物由来発酵代謝物の調製
製造例1の(ii)と同様の方法で、酵母抽出物溶液を得た。
(iii)組成物の調製
サクラ抽出物溶液と酵母抽出物溶液を、1:1の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.38%)
(i)サクラの抽出物の調製
サトザクラに代えてソメイヨシノを用いるほかは、製造例2の(i)と同様の方法により、褐色透明のサクラ抽出物溶液1355gを得た(固形分濃度0.40%)。
(ii)微生物由来発酵代謝物の調製
製造例2の(ii)と同様の方法により酒粕抽出物溶液を得た。
(iii)組成物の調製
サクラの抽出物溶液と酒粕抽出物溶液を、1:9の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.31%)。
(i)サクラの抽出物の調製
サトザクラに代えてソメイヨシノを用いるほかは、製造例3の(i)と同様の方法により、褐色透明のサクラ抽出物溶液1340gを得た(固形分濃度0.34%)。
(ii)微生物由来発酵代謝物の調製
製造例3の(iii)と同様の方法にて、乳酸菌抽出物溶液を得た。
(iii)組成物の調製
サクラ抽出物溶液と乳酸菌抽出物溶液を、1:2の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.48%)
(i)サクラの抽出物の調製
製造例2の(i)と同様の方法により、サクラ抽出物溶液を得た。
(ii)微生物由来発酵代謝物の調製
精白米50gを蒸して冷却した後、アスペルギルス オリゼー(Aspergillus oryzae)を添加し、これを30℃の温度条件で米麹を作製した。これに、別に蒸した精白米50gと殺菌した精製水200gを加え、さらに予め培養したサッカロミセス セレビシエ(Saccharomyces cerevisiae)培養液を添加して、30℃で発酵を行った。発酵終了後、この米発酵物溶液を90℃で1時間、加熱殺菌処理した後、室温に戻し、濾過をして淡黄色透明の米発酵物溶液126gを得た(固形分濃度4.00%)。
(iii)組成物の調製
サクラの抽出物と米発酵物溶液を、1:9の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.30%)。
(i)サクラの抽出物の調製
製造例3(i)と同様の方法により、サクラ抽出物溶液を得た。
(ii)微生物由来発酵代謝物の調製
米麹100gに予め滅菌しておいた精製水を300g加え、35℃で静置した。その後、これに別に蒸した精白米100gと予め培養したサッカロミセス セレビシエ(Saccharomyces cerevisiae)培養液を添加して30℃で発酵を行った。発酵終了後、この米発酵物溶液を90℃で1時間、加熱殺菌処理した後、室温に戻し、濾過をして淡黄色透明の米発酵物溶液183gを得た(固形分濃度3.93%)。
(iii)組成物の調製
サクラの抽出物と酒粕抽出物を、1:9の混合比で混合後、不溶物を濾過し、褐色透明の抽出物混合溶液を得た(固形分濃度0.31%)。
製造例1の(i)と同様の方法によりサクラ抽出物溶液を調製し、これを比較試料(1)とした。
製造例2の(ii)と同様の方法により酒粕抽出物溶液を調製し、これを比較試料(2)とした。
[A成分] 部
オリーブ油 1.0
ポリオキシエチレン(5.5)セチルアルコール 5.0
ブチルパラベン 0.1
[B成分]
製造例1の組成物(1) 5.0
エタノール 5.0
グリセリン 5.0
1,3−ブチレングリコール 5.0
水酸化カリウム 適量
精製水 全量が100部となる量
[C成分]
香料 適量
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例2の組成物(2)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例3の組成物(3)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例4の組成物(4)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例5の組成物(5)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例6の組成物(6)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例7の組成物(7)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例8の組成物(8)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例9の組成物(9)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例10の組成物(10)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
処方例1のB成分中、製造例1の組成物(1)に代えて、製造例11の組成物(10)5.0部を用いるほかは処方例1と同様にして化粧水を得た。
[A成分] 部
流動パラフィン 6.0
ヘキサラン 4.0
ホホバ油 1.0
ポリオキシエチレン(20)ソルビタンモノステアレート 2.0
大豆レシチン 1.5
[B成分]
製造例2の組成物(2) 3.0
L−アスコルビン酸−2−グルコシド 2.0
水酸化カリウム 0.5
グリセリン 3.0
1,3−ブチレングリコール 2.0
カルボキシメチルセルロース 0.3
ヒアルロン酸ナトリウム 0.01
精製水 全量が100部となる量
[C成分]
香料 適量
処方例12のB成分中、L−アスコルビン酸−2−グルコシド2.0部及び水酸化カリウム0.5部に代えてトラネキサム酸2.0部を用いるほかは処方例12と同様にして乳液を得た。
処方例12のB成分中、L−アスコルビン酸−2−グルコシド2.0部及び水酸化カリウム0.5部に代えてアルブチン3.0部を用いるほかは処方例12と同様にして乳液を得た。
処方例12のB成分中、L−アスコルビン酸−2−グルコシド2.0部及び水酸化カリウム0.5部に代えてニコチン酸アミド5.0部を用いるほかは処方例12と同様にして乳液を得た。
[成分] 部
エタノール 2.0
グリセリン 5.0
1,3−ブチレングリコール 5.0
メチルパラベン 0.1
ヒアルロン酸 0.1
製造例5の組成物(5) 5.0
クエン酸 0.3
クエン酸ナトリウム 0.6
精製水 全量が100部となる量
[成分] 部
製造例6の組成物(6) 10.0
エタノール 10.0
グリセリン 3.0
1、3−ブチレングリコール 2.0
メチルパラベン 0.2
クエン酸 0.1
クエン酸ナトリウム 0.3
カルボキシビニルポリマー 0.1
キサンタンガム 0.1
香料 適量
水酸化カリウム 適量
精製水 全量が100部となる量
上記の成分を混合してローションを得た。
[A成分] 部
ステアリン酸 2.4
モノステアリン酸プロピレングリコール 2.0
セトステアリルアルコール 0.2
液状ラノリン 2.0
流動パラフィン 3.0
ミリスチン酸イソプロピル 8.5
プロピルパラベン 0.05
[B成分]
製造例7の組成物(7) 5.0
カルボキシメチルセルロースナトリウム 0.2
ベントナイト 0.5
プロピレングリコール 4.0
トリエタノールアミン 1.1
メチルパラベン 0.1
精製水 全量が100部となる量
[C成分]
酸化チタン 8.0
タルク 4.0
着色顔料 適量
[A成分] 部
N−ヤシ油脂肪酸メチルタウリンナトリウム 10.0
ポリオキシエチレン(3)アルキルエーテル硫酸ナトリウム 20.0
ラウリルジメチルアミノ酢酸ベタイン 10.0
ヤシ油脂肪酸ジエタノールアミド 4.0
メチルパラベン 0.1
[B成分]
クエン酸 0.1
製造例10の組成物(10) 2.0
1,3−ブチレングリコー ル 2.0
精製水 全量が100部となる量
[A成分] 部
ポリオキシエチレン(10)硬化ヒマシ油 1.0
塩化ジステアリルジメチルアンモニウム 1.5
塩化ステアリルトリメチルアンモニウム 2.0
2−エチルヘキサン酸グリセリル 1.0
セタノール 3.2
ステアリルアルコール 1.0
メチルパラベン 0.1
[B成分]
製造例1の組成物(1) 2.0
1,3−ブチレングリコール 5.0
精製水 全量が100部となる量
[成分] 部
グリチルリチン酸ジカリウム 0.1
モノニトログアヤコールナトリウム 0.02
塩酸ピリドキシン 0.03
アデノシン 1.0
製造例2の組成物(2) 1.0
トリメチルグリシン 0.5
乳酸 0.2
1,3−ブチレングリコール 10.0
フェノキシエタノール 0.2
ポリオキシエチレン硬化ヒマシ油 0.4
タマサキツヅラフジ根エキス 0.3
オタネニンジンエキス 0.3
L−アルギニン 適量
精製水 全量が100部となる量
ヒト表皮細胞NHEKを、HuMedia KG2培地(クラボウ社製)を入れた96穴マイクロプレートに5×103個/穴播種し、37℃、5.0%CO2の条件下に1日間プレ培養した後、製造例1〜10の組成物(1)〜(10)のそれぞれを試料溶液として含む培地(Humedia KG2)をプレ培養液に添加し、同条件でさらに2日間培養した。ここで、試料溶液の濃度は、追添加する培地全量に対する溶液としての終濃度が1.0%、2.0%となるように調製した。プレ培養後、培地を除去し、0.03%のMTTを添加して37℃に1時間保持した後、生成したホルマザンをイソプロパノールで抽出し、マイクロプレートリーダー(Model 680、バイオラッド社製)を用いて波長570−630nmでMTT値を測定した。また、試料無添加の場合(Control)についても上記と同様の操作を行った。そして、試料無添加時のMTT値を100としたときの各試料添加時のMTT値の相対値を求め、表皮細胞MTT活性率(%)とした。また、試験系が正常に機能しているかを確認するために、試料溶液の代わりに陽性対照として100mMのグルコースを添加した場合についても、同様の試験を行った。
[表1]
DPPH(1,1−ジフェニル−2−ピクリルヒドラジル)2.4部をエタノール20部に溶解し、これに精製水20部を加えてDPPH溶液を調製した。このDPPH溶液24部に対して、18v/v%エタノール溶液を19.2部、2M酢酸−酢酸ナトリウム緩衝液(pH5.5)を4.8部加えて、DPPH添加溶液として調製した。また、抽出液そのものの色調が試験に及ぼす影響を差し引くため、DPPH溶液の代わりに50v/v%エタノール溶液を用いて、18v/v%エタノール溶液と2M酢酸−酢酸ナトリウム緩衝液を混合した液を対照液とした。次に、製造例1〜10の組成物(1)〜(10)及び比較試料(1)〜(2)を精製水でそれぞれ希釈して12種の試料溶液を調製した。なお、各組成物は、試料溶液全量に対する溶液としての終濃度がそれぞれ1.0%、2.0%となるように精製水で希釈した。この試料溶液とDPPH添加溶液又は対照液とを1:3の割合で混合し、室温で10分静置後、各試料溶液をDPPH添加溶液と混合した場合の550nmにおける吸光度と、各試料溶液を対照液と混合した場合の550nmにおける吸光度との差を測定し、DPPHラジカルの残存量を確認した。また、試料無添加の場合(Control)についても上記と同様の操作を行い、試料無添加時のDPPHラジカル残存率を100としたときの各試料添加時のDPPHラジカル残存率の相対値を求めた。また、試験系が正常に機能しているかを確認するために、試料溶液の代わりに陽性対照として水溶性ビタミンE[Trolox](終濃度25μM)を添加した場合についても、同様の試験を行った。
[表2]
1Mトリス−塩酸緩衝液0.15mL、1mMエチレンジアミン四酢酸・二ナトリウム塩溶液0.30mL、1mMキサンチン溶液0.30mL、0.75mMニトロブル-テトラゾリウム溶液0.20mL、製造例1〜10の組成物(1)〜(10)及び比較試料(1)〜(2)の各0.10mLと精製水1.90mLとを混合して12種の試験溶液を調製した。また、試験溶液に代えて精製水2.00mLを用いる他は上記試験溶液と同様の組成からなる混合液(コントロール[Control])を調製した。さらに、試料溶液(0.10mL)に代えて、0.875Unit/mLのスーパーオキシドジスムターゼ(SOD)溶液0.10mLを用いる他は上記試験溶液と同様の組成からなる混合液(陽性対照液)を調製した。上記試験溶液をそれぞれ37℃でインキュベートした後、これに1Unit/mLキサンチンオキシダーゼ溶液0.05mLを添加し、一定時間経過後(5分)、各試験溶液の570nmでの吸光度(被験液中のスーパーオキシドアニオン量の指標)を測定した。測定結果は、試料無添加(Control)の混合液の吸光度を100とした時の各試験溶液及び陽性対照液の吸光度を相対値で示した。
[表3]
正常ヒト表皮細胞を、増殖添加剤含有HuMedia KG2培地(クラボウ社製)にて6×105個/mLに調製し、φ6cmシャーレに1mLを播種して、5%CO2、飽和水蒸気下、37℃で培養した。24時間培養後、さらに、製造例1〜3及び10の組成物(1)〜(3)及び(10)を試料溶液として含んだ同培地(培地全量に対して溶液としての終濃度が1.0%となるように試料溶液が含まれるもの)を追添加して培養した。また、試料無添加(Control)の場合についても上記と同様の操作を行った。24時間培養後、それぞれの試験区の細胞をTrizol試薬(Invitrogen社製)1mLにより回収した。回収した細胞に対してクロロホルム(和光純薬工業社製)200μLを添加して撹拌混合し、遠心分離機(TOMY社製/MX-160)で15,000rpm、4℃の条件下で15分間遠心分離した後、水層のみを400μL分取した。回収した水層にイソプロパノール(和光純薬工業社製)500μLを添加して撹拌混合し、15,000rpm、4℃の条件下で15分間遠心分離してtotal RNAの沈殿物を得た。total RNAに75%エタノールを1mL添加して撹拌して洗浄し、15,000rpm、4℃条件下で15分間遠心分離して沈殿を回収した。回収したtotal RNAを所定のキット(PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time)[タカラバイオ社製])を用いて逆転写反応し、cDNAを合成した。合成したcDNAをサンプルとして、Thermal Cycler Dice(登録商標)Real Time System Single(タカラバイオ社製)、及びSYBR(登録商標)Premix Ex TaqTM II(Perfect Real Time)[タカラバイオ社製]を用いて、XVII型コラーゲン遺伝子の発現と、内部標準物質βアクチン遺伝子の発現の検出を行った。ここで、βアクチンは、ハウスキーピング遺伝子(多くの組織や細胞中に共通して一定量発現する遺伝子であって、常に発現され,細胞の維持,増殖に不可欠な遺伝子である)の一つであり、発現量が常に一定とされていることから、PCRの実験では内部標準として用いられるものである。試験結果は、βアクチン遺伝子の発現量を一定とした場合の、それぞれの試験区でのXVII型コラーゲン遺伝子の発現量を比較した。本試験系においては、コントロール区のそれぞれの遺伝子の発現量を100としたときの試験区での当該遺伝子の発現量の相対値を求めた。
[表4]
正常ヒト皮膚由来表皮細胞(NHEK)をHuMedia KG2培地(クラボウ社製)を入れた96穴マイクロプレートに8×103個/穴播種し、37℃,5.0%CO2の条件下に1日間プレ培養した後、試料溶液を含む同培地(培地全量に対して溶液としての終濃度が1.0%,2.0%となるように試料溶液が含まれるもの)を追添加し、同条件でさらに2日間培養した。その後XVII型コラーゲン抗体を用いた免疫的検出を行った。すなわち、PBS(-)洗浄後、15%中性緩衝ホルマリン液を用いて細胞を30分処理して固定、0.5%Triton X-100溶液で1時間浸透処理、5倍希釈ブロッキングワンP(ナカライテスク社)溶液で2時間処理によるブロッキングを行った後、XVII型コラーゲン抗体を添加し、4℃で一昼夜静置した。その後PBS(-)洗浄し、蛍光ラベルした二次抗体を添加してさらに暗所で一定時間静置した。そのPBS(-)後洗浄し、蛍光強度の測定を行った。まず、二次抗体の蛍光ラベル(Alexa Fluor488)をEx=485nm、Em=520nmで測定し[蛍光マイクロプレートリーダー(フルオロスキャンアセント、Thermo Fisher Scientific社製)]、その後、Hoechst33342によるDNA染色を行い、Ex=355nm、Em=460nmの測定を行った。それぞれの試験区のAlexa Fluor488の蛍光強度をHoechst33342の蛍光強度で割ることで、XVII型コラーゲンの生成度合いを求めた。また、試料無添加の場合(Control)についても上記と同様の操作を行い、ここに得られたXVII型コラーゲン生成度合いに対する各試料添加時のXVII型コラーゲン生成度合いの相対値を求め、XVII型コラーゲン生成量(%)とした。
[表5]
培養B16マウスメラノーマ細胞B16−F10を、24穴マイクロプレートに2.4×104個/穴播種し、10%FBS含有RPMI1640培地中、37℃、5.0%CO2の条件下に1日間プレ培養した後、同培地で、試料溶液を溶液として終濃度が1.0%、2.0%となるように希釈した溶液と終濃度1mMになるように調整したテオフィリン含有培地を添加し、同条件で2日間培養した。次に培養液を除去し、1N NaOH/10%ジメチルスルフォキシド溶液を1穴あたり200μL添加し、シールして50℃、2時間インキュベートして細胞を溶解させた。この溶液100μLを別の96穴マイクロプレートに移し、マイクロプレートリーダー(Model 680、バイオラッド社製)を用い、波長490nmでメラニン値を測定した。一方同じ細胞を溶解させた溶液を5μL別の96穴マイクロプレートに移し、さらに精製水で5倍希釈したDye Reagent Concentrate(バイオラッド社)溶液を200μL添加し、 マイクロプレートリーダー(Model 680、バイオラッド社製)を用い、波長570nmの吸光度を測定した。別で既知の量の牛血清アルブミン(Sigma社製)を段階希釈し、同様に操作して得られた検量線から、アルブミン当量のタンパク質量を計測した。得られた吸光度をタンパク質量で除算して、タンパク質あたりのメラニン量を求めた。また、試料無添加の場合(Control)についても上記と同様の操作を行い、ここに得られたタンパク質あたりのメラニン量に対する各試料添加時のタンパク質あたりのメラニン量の相対値を求め、メラニン合成率(%)とした。なお、比較のため、試料溶液の代わりに、2mMのコウジ酸を添加した場合(陽性対照)についても同様の試験を行った。
[表6]
以下、本発明に係る組成物の脂肪蓄積の抑制効果を、脂肪細胞内の脂肪滴周辺に存在するタンパク質[ペリリピン1(perilipin 1)]の合成抑制効果により評価する。このペリリピン1が減少すると、脂肪滴数の減少及び脂肪滴サイズの縮小が生じることが知られていることから、ペリリピン1の合成抑制効果を評価する。
マウス線維芽細胞(3T3-L1)を、10%FBS含有ダルベッコ変法イーグル最少必須培地(DMEM:日水製薬株式会社)を入れた96穴マイクロプレートに1.5×104個/穴播種し、37℃,5.0%CO2の条件下に3日間プレ培養した後、分化誘導培地(10%FBS、0.5mM3-イソブチル-1-メチルキサンチン(IBMX)、0.25μMデキサメタソン(DEX)及び1.1μg/mLインスリンを混合したDMEM)を添加後、さらに2日間培養した。その後、試料溶液を含む同培地(培地全量に対して溶液としての終濃度が1.0%,2.0%となるように試料溶液が含まれるもの)を追添加し、同条件でさらに5日間培養した。その後、ペリリピン1(perilipin1)抗体を用いた免疫的検出を行った。すなわち、PBS(-)洗浄後、細胞を10%中性緩衝ホルマリン液にて30分処理して固定、0.5%Triton X-100溶液で1時間浸透処理、5倍希釈ブロッキングワンP(ナカライテスク社)溶液で2時間処理によるブロッキングを行った後、perilipin1抗体を添加し、室温で1時間静置した。その後PBS(-)洗浄し、蛍光ラベルした二次抗体を添加してさらに暗所で一定時間静置した。その後PBS(-)洗浄し、蛍光強度の測定を行った。まず、二次抗体の蛍光ラベル(Alexa Fluor488)をEx=485nm、Em=520nmで測定し[蛍光マイクロプレートリーダー(フルオロスキャンアセント、Thermo Fisher Scientific社製)]、その後、Hoechst33342によるDNA染色を行い、Ex=355nm、Em=460nmの測定を行った。それぞれの試験区のAlexa Fluor488の蛍光強度をHoechst33342の蛍光強度で割ることで、perilipin1の生成度合いを求めた。また、試料無添加の場合(Control)についても上記と同様の操作を行い、ここに得られたperilipin1生成度合いに対する各試料添加時のperilipin1生成度合いの相対値を求め、perilipin1合成率(%)とした。
[表7]
Claims (3)
- バラ科(Rosaceae)サクラ属(Prunus)に属するサクラの抽出物と、酵母、麹菌及び乳酸菌のいずれか1種又は2種以上の抽出物又は酵母、麹菌及び乳酸菌のいずれか1種又は2種以上の微生物により産生される微生物由来発酵代謝物とを含有する組成物。
- 請求項1に記載の組成物を含有する皮膚外用剤。
- 請求項1に記載の組成物を含有する飲食品。
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